Methods for using tetanus toxin for beneficial purposes in animals (mammals)

ABSTRACT

Methods of using tetanus toxin to modulate or control neural functions or nonneural cellular activities at selected sites in animals, particularly in mammals, and more particularly in humans, are provided. Pharmaceutical formulations to modulate neural functions or non-neural cellular activities of an animal at selected sites in animals, particularly in mammals, and more particularly in humans are also provided. Uses of tetanus toxin in preparation of medicaments for methods of treating clinical disorders or symptoms of animals, particularly mammals and more particularly humans are also provided.

FIELD OF INVENTION

The invention relates broadly to methods of modulating a neural functionof an animal, including a mammal, at a selected site. The invention alsorelates broadly to methods of modulating other nonneural cellularactivity of an animal at a selected site. The invention also encompassespharmaceutical formulations for modulating a neural function or anon-neural cellular function of an animal at a selected site. Theinvention also relates to the use of tetanus toxin in the preparation ofmedicaments for methods of treating clinical disorders or symptoms of ananimal.

BACKGROUND OF THE INVENTION

The Clostridial neurotoxins are the most potent toxins known to man.When Clostridium botulinum bacteria are ingested orally they producebotulinum toxin (BT). BT is absorbed from the gastrointestinal tract andis transported by the circulatory system to muscles throughout the body.The BT binds to and blocks neuromuscular transmission from motor neuronscausing a fatal paralysis known as botulism.

An unusual attribute of the BT is that its action lasts for months butthe patient completely recovers. As a result of this unique attribute,BT has many clinical uses. At present, the local injection of smalldoses of BT is used to decrease or block muscle activity in a widevariety of clinical motor disorders. More recently, the use of BT hasbeen extended to block autonomic nerves that use the sameneurotransmitter used in neuromuscular transmission, namely,acetylcholine.

The other class of Clostridial bacteria is Clostridia tetani. Thesebacteria infect wounds and produce tetanus toxin. Tetanus toxin (TT) isreleased from the site of the infection and is distributed by thecirculatory system to motor neurons throughout the body. Instead ofacting on the motor neurons directly, the tetanus toxin is transportedto the central nervous system where it blocks neurons that normallyinhibit motor neuron activity. The result is a gradually increasing tonein affected muscles that culminates in a widespread spasm of musclesthroughout the body. The resulting spastic paralysis is often fatal withdeath resulting from respiratory depression or circulatory collapse.

Tetanus has been recognized as a disorder since antiquity and it isstill common throughout the world. Many countries routinely vaccinatechildren with tetanus toxoid, an attenuated form of the toxin that isexposed to formaldehyde to remove its biological activity whileretaining its antigenicity. Tetanus toxoid is the largest biologicproduct in the pharmaceutical industry.

The action of the tetanus toxin lasts from weeks to months. Once TTenters into and blocks neurotransmission from neuron synapses theprocess is irreversible. Recovery of function requires the growth of anew process from the neuron that eventually reconnects to the motorneuron and restores the inhibitory activity back to normal levels. Thetime required for this recovery varies from weeks to up to five months(Struppler, A., et al. Arch Neurol, 8, 162-1782, (1963)).

The extremely broad range of TT actions allows it to either excite orinhibit practically any part of the nervous system for prolonged periodsof time with a single injection. Since the nervous system closelymonitors and controls nearly every organ and physiological function ithas been unexpectedly found that TT can have extensive beneficialutility for the treatment or amelioration of a wide variety of clinicaldisorders.

Tetanus is a systemic intoxication by the tetanus toxin which ischaracterized by progressive spastic contraction of the skeletal musclesand overactivity of the autonomic nervous system that is often fatal.

Other than the systemic disorder three lesser known variants or tetanusare known. These are neonatal, cephalic and local tetanus. Neonataltetanus is a fatal intoxication of newborn babies that is manifest as asystemic flaccid paralysis. Cephalic tetanus occurs on the face andcombines a localized paralysis, most often of the facial nerve, with asurrounding area of muscle spasm (Dastur, F. D., et al., Journal OfNeurology, Neurosurgery And Psychiatry 40(8), 782-6 (1977)). Localtetanus is an isolated spasm of a muscle group or limb that may progressto systemic tetanus or resolve over weeks to months (Johns HopkinsMedical Journal, 149(2) 84-8, (1981); Jain, S., et al., Journal OfNeurology, 228(4), 289-93, (1982)).

The tetanus toxin has some unique properties that have made it perhapsthe most studied of all biological toxins. For example, the tetanustoxin binds to all types of neurons. Although its primary affinity is tobind to motor neurons, TT also binds to neurons of the autonomic nervoussystem and sensory neurons (Stockel, K., et al., Brain Research, 99,1-16 (1975)). In contrast, the botulinum toxins principally bind tomotor neurons.

Tetanus toxin requires multiple specific steps to cause its effects inneurons. These steps include (i) peripheral binding; (ii)internalization; (iii) retrograde transport; (iv) central binding; and(v) transmembrane internalization.

In peripheral binding the toxin binds to the surface of the cell. TTbinds to the presynaptic membrane of practically all neurons. Inaddition it also binds to the membrane of the neuron's axon. Thereceptors to which TT binds are a class of molecules known asgangliosides. BT also binds to gangliosides, however BT appears to bindprincipally to those on the presynaptic membrane of cholinergic neurons,whereas TT binds to the pre-synaptic membrane of most if not allneurons.

In the internalization step the toxin is brought into the cell. TT isbrought into the neuron by the process of forming a vesicle. While TTremains inside the vesicle, although physically inside the neuron, thetoxin is separated from the cytoplasm of the neuron by a membrane. Incontrast, BT is thought to require a second molecule on the presynapticmembrane to bind to before being internalized. After BT binds to thesecond molecule it passes through the cell membrane directly into thecytoplasm, which is why it exerts its effect at the peripheralpresynaptic membrane.

During the retrograde transport the vesicles containing TT aretransported to the cell body in the central nervous system. The vesiclethen fuses with the cell membrane of the cell body or its dendritesthereby depositing TT into the extracellular space between the motorneuron on the processes of other neurons synapsing onto the motor neuron(Hilbig, G., K. O. Räker, et al., Naunyn-Schmiedebergs Archives OfPharmacology, 307(3), 287-90, 1979.

In the central binding step TT can bind to all neurons. However, TT hasa much greater affinity for the inhibitory neurons. At lowconcentrations, tetanus has greater affinity for the neurons that usethe inhibitory neurotransmitters GABA and glycine (Montecucco, C. etal., Q Rev Biophys, 28(4), 423-72, (1995)). At higher concentrations, itblocks all neurotransmitters. Finally, tetanus toxin has a local effecton axons that causes a local block of the propagation of actionpotentials. The mechanism for this is unknown but the result is similarto the action of a local anesthetic.

During the transmembrane internalization, once the toxin binds to asecond neuron it is internalized and produces its toxic effect.

The primary mechanism of action of TT is to block the release ofvesicles from a cell. In neurons these vesicles containneurotransmitters. The proteins that are involved in the attachment of avesicle to the inner membrane of a cell are the SNARE (synaptosomeassociate protein receptor) family of proteins. These proteins are partof the mechanism by which intracellular vesicles dock to cell membranesand release their contents. Specifically, tetanus toxin cleaves VAMP(vesicle associated membrane protein). Botulinum toxins A and E c1cavcSNAP-25; and botulinum toxin C cleaves SNAP-25 and syntaxin; tetanustoxin and botulinum neurotoxins type B, D, F and G cleave VAMP, anintegral protein of the neurotransmitter containing synaptic vesicles.

The mechanism of vesicle release is common to all cells from yeast tothe cells of humans. The TT molecule is composed of a heavy chain thatis responsible for its specific binding and transport properties, and alight chain that actually performs the catalytic action on the VAMPprotein. There are a few non-neuronal cells in which TT is capable ofentering and performing its action and these will be discussed in theexamples.

Multiple experiments have shown that even if TT is incapable of bindingand entering into a type of cell it can be inserted by a variety ofmechanisms. Once inside the cell TT cleaves VAMP and disables vesiclerelease. Different cells use the mechanism to secrete hormones,neuropeptides, lysozyme proteins, and other substances. Whateverspecific substance is secreted by the cell it will be blocked by TT ifsecretion requires the use of VAMP protein.

Cells can be made susceptible to TT by placing gangliosides onto theexternal surface of the cell membrane. Another manner of inserting TTinto cells is by chemically combining the TT, or at a minimum its lightchain, with a second molecule that is capable of binding to the cell. Inaddition the TT can be inserted by micro injection using micropipettes,pressure injection, by incorporation into lysosomes, or by temporarilymaking the cell membrane permeable to TT.

Motor neurons are the primary target of TT toxicity. Motor neurons referto cholinergic neurons that innervate the large extrafusal muscle fibersof skeletal muscle. Within skeletal muscle are smaller intrafusal musclefibers and these are innervated by a smaller cholinergic motor neuroncalled a gamma neuron. These also are intoxicated by TT. Finally, smoothmuscle is also innervated by cholinergic neurons. Although the vastmajority of these neurons are cholinergic and are formally consideredpart of the autonomic nervous system they are sometimes grouped with theother motor neurons for discussion because they have certainbiologically similar properties. TT has been shown to bind to andintoxicate the motor neurons of smooth muscles in the same manner as itdoes to striated muscle fibers.

It should be noted that at high doses TT can cause a flaccid paralysisby blocking motor neuron activity both in the periphery, at theneuromuscular junction, and centrally, by blocking all afferent inputfrom both excitatory and inhibitory axons. Which of the two areaspredominate in a given case of intoxication is dependent on where the TTinfection is and how it progresses.

In cephalic tetanus all three actions of TT can occur together. At thesite of infection the muscle exhibits a flaccid paralysis as TT levelsare high and the neuromuscular synapses are blocked directly. Inaddition high levels of the toxin are transported back to the brainstemto block central nervous system input. However at variable distancessurrounding the site of infection the concentration of TT falls untilareas are seen in which muscles are in spasm (Dastur, F. D., et al.,Journal Of Neurology, Neurosurgery And Psychiatry 40(8), 782-6 (1977)).

Tetanus toxin can be measured by weight or, more commonly, by biologicalassay. The effective dose of tetanus is measured in units, the amount oftetanus toxin that is lethal to 50% of mice when injectedsubcutaneously. A unit of tetanus toxin may range between 0.1 to 100 ngof toxin per kg of mouse body weight. In the mouse hemi diaphragm assay,a 500 times higher dose of tetanus toxin is needed to cause a flaccidparalysis (Bigalke, H. et al., Naunyn Schmiedebergs Arch Pharmacol,312(3), 255-63, (1980)). Therefore at equivalent weights tetanus toxinwould be expected to cause a spastic paralysis while botulinum toxincauses a flaccid paralysis.

Tetanus toxin has the same effects on autonomic neurons as it does onmotor neurons (Abboud, F. M., Hypertension, 4 (3 Pt 2), 208-25, (1982)).In systemic tetanus excitation of the autonomic nervous system isprominent and manifest by such symptoms as high blood pressure (Toriya,Y., T. et al. Endodontics And Dental Traumatology, 13 (1), 6-12,(1997)), erratic changes in blood pressure, high fever, and profusesweating. Therefore, low concentrations result in increased autonomictone with physiological changes resulting in all organs affected.

The autonomic nervous system is divided into the parasympathetic systemwhich uses acetylcholine as its neurotransmitter and the sympatheticsystems which uses epinephrine as the neurotransmitter. In both theparasympathetic and sympathetic systems neurons do not reach the entiredistance from the central nervous system to the peripheral organ.Instead the distance requires two neurons with a synapse somewhere inthe periphery. In the sympathetic system these are grouped together in alimited number of large ganglia located near the spinal column. In theparasympathetic organ the synapse is usually located in a smallerganglia near the target organ. In both systems the neurotransmitter usedin the ganglia synapses is always acetylcholine.

Although the peripheral neurons of the sympathetic nervous system mayall use norepinephrine as their neurotransmitter, the response of thetarget organ cells is dependent on the type of receptor. There are threetypes of adrenergic receptors: alpha (smooth muscle contraction), beta1(cardiac acceleration and fatty acid mobilization) and beta 2 (smoothmuscle relaxation). Note that the exact effect of norepinephrine may beentirely opposite on different muscles based on the type of receptorsfound on the surface. Much of this information is known for most organsthat are clinically relevant.

The presence of ganglia and multiple neurotransmitters increases thecomplexity of the autonomic system relative to the motor system. Anorgan is usually innervated by both parasympathetic and sympatheticneurons and they usually have opposite actions on the target. Thereforean understanding of the anatomy and physiology of the target organ isimportant in planning the location of a TT injection so that the desiredeffect is to be achieved.

Sensory neurons can also be blocked by tetanus toxin. Sensoryneuropathies are part of the symptom complex of clinical tetanus. Oneaspect of Clostridial tetanus infection is that pain and inflammation atthe site of infection are much lower then would be expected in suchserious infections. Therefore, it is believed that T′ blocks sensoryneurons.

In addition to neurotransmitters, which are usually used to directlycommunicate with other neurons through synaptic connections, neuronsrelease neuropeptides from motor, sensory and autonomic nerves (SP,substance P; NKA, neurokinin A; CGRP, calcitonin gene-related peptide;NPY, neuropeptide Y, interleukins and growth factors). Theseneuropeptides have many different effects but one of the most importantis vasodilatation and inflammation. (Bigalke, H. et al, NaunynSchmiedebergs Arch Pharmacol, 312(3), 255-63, (1980)). Theseneuropeptides are released by the same vesicle mechanism asneurotransmitters and therefore can be blocked by TT.

The SNARE proteins and the vesicle release mechanism are used by cellsfor purposes other then the release of neurotransmitters. In fact, therelease of practically all cellular secretions depends on thismechanism. These include the release of hormones, enzymes, andinflammatory modulators, mucus secretions from respiratory, digestiveand urinary glands, and inflammatory modulators from nerves and whiteblood cells (Alexander, E. A. et al., American Journal of Physiology,273 (6 Pt 2), F1054-7 (1997)). Cells known to internalize tetanus toxininclude macrophages, endocrine cells, and renal cells (Huet de la Tour.E., et al., Journal Of The Neurological Sciences, 40(2-3), 123-31,(1979)).

In addition to their effect on SNARE proteins, Clostridial toxins havebeen shown to interfere with other cell activities. For example, it canprevent actin molecules from forming into filaments. Actin is the maincellular skeleton protein involved in cell shape and movement. Thisaction can block the contraction of muscle cells as well as stop themigration of white blood cells and possibly malignant cells also. Thetoxins also interfere with cell signaling. Specifically, receptors on acell's surface respond to specific molecules by promoting a cascade ofsecondary proteins that in turn result in a variety of cell functionsfrom changes in morphology to secretion.

In “Ophthalmic and Reconstructive Surgery,” 16 (2), 101-13, (2000),Fezza J. P. et al. disclose the use of tetanus toxin to cause localizedorbiculari oculi weakness without producing systemic tetany in immunizedrabbits. Potential uses of tetanus toxin in treatment of blepharospasmand hemifacial spasm are suggested without provisions of any detailedinformation regarding dosage or other description useful to one skilledin the art seeking to use the tetanus toxin to treat these conditions.

U.S. Pat. No. 5,989,545 to Foster et al. describes the use of the lightchain of a clostridial neurotoxin by itself or linked to other moietiesas a pharmaceutical for the treatment of pain. Foster et al. do notdisclose the use of the entire molecule of tetanus toxin.

U.S. Pat. No. 5,714,468 to Binder describes the use of a fragment oftetanus toxin to reduce pain in migraine headaches. U.S. Pat. No.5,670,484 to Binder discloses a method for treatment of cutaneouscell-proliferative disorders with Botulinum toxin A and tetanus toxin.In both patents, Binder uses the same TT dosages as are used for BT.Moreover, he discourages the use of TT for beneficial purposes becausebe found that TT to be too toxic at the dosages disclosed in hispatents.

U.S. Pat. No. 5,766,605 to Sanders et al. describes the control ofautonomic nerve function in a mammal by administering to the mammal atherapeutically effective amount of Botulinum toxin. There is nodisclosure of tetanus toxin.

Despite the apparent effects of neurotoxins on motor, autonomic andsensory neurons, the use of such toxins, and especially tetanus toxin inanimals, including humans, has been limited and has never been used forclinical applications. Thus, there remains a need in the medical art formethods of treating patients with tetanus toxin that can cause anincrease or decrease in neural activity at selected sites of thepatient. Similarly, there is still a need in the medical arts formethods using tetanus toxin to treat clinical disorders caused byimproper cellular activity, such as inflammatory conditions. Further,there remains a need for pharmaceutical formulations that can bedelivered to a patient to achieve clinically beneficial results or treatcertain dysfunctions, while eliminating or minimizing dependence,tolerance, and side effects associated with more conventional drugs.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a method of using tetanustoxin to achieve beneficial effects in animals, particularly in mammalsand more particularly in humans.

It is an object of the invention to provide a treatment of neuromusculardysfunctions in animals, particularly in mammals and more particularlyin humans.

It is an object of the invention to provide a treatment of autonomicnerve dysfunctions in animals, particularly in mammals and moreparticularly in humans.

It is another object of the invention to control sensory functions inparticularly animals, particularly in mammals and more particularly inhumans.

It is an object of the invention to modulate or control neural functionsin animals, particularly in mammals and more particularly in humans.

It is another object of the invention to use tetanus toxin modulatenon-neural cellular activities of cells in animals, particularly inmammals and more particularly in humans.

SUMMARY OF THE INVENTION

In view of the above objects and others, the present invention isdirected in part to a method of modulating a neural function of ananimal at a selected site affected by target neurons, the methodincluding administering a therapeutically effective amount of tetanustoxin to the selected site of the animal such that the tetanus toxinreversibly modulates the activity of the target neurons.

In one aspect of the present invention, a therapeutically effectiveamount of tetanus toxin is sufficient to cause decrease in neuralactivity or reversible inhibitory response of the neural activity at theselected site.

In another aspect of the present invention, the therapeuticallyeffective amount of tetanus toxin is sufficient to cause increase intarget neuron activity or an excitatory response of the neural activityat the selected site.

In another embodiment, the present invention is further directed to amethod for decreasing the activity of a nerve function in an animalincluding administering to a selected site affecting target neurons ofan animal an amount of tetanus toxin sufficient to cause a denervationof the target neurons, wherein the denervation results in a reversibleinhibitory response of the nerve function at the selected siteinnervated by the target neurons.

In yet another embodiment, the present invention is directed to a methodfor increasing the activity of a nerve function in an animal comprisingadministering to a selected site affecting target neurons of an animalan amount of tetanus toxin sufficient to cause an excitatory response ofthe nerve function at the selected site innervated by the targetneurons.

In another aspect, the invention is related to a method for controllingneural function in animals, particularly mammals, and more particularlyhumans, comprising administering to a selected site a therapeuticallyeffective amount of tetanus toxin to control the neural function.

In certain embodiments of the present invention including each of theforegoing methods, the decrease or increase in neural activity occursover a period of time from about one hour to about one year. In certainpreferred embodiments, the decrease or increase in neural activityoccurs over a period of time from about one week to about four months.

In certain embodiments of each of the foregoing methods, each of theforegoing methods includes: (i) determining the level of antibodies oftetanus toxin present in blood plasma of the animal prior toadministering of any tetanus toxin; and (ii) immunizing the animal whenthe level of tetanus toxin is below 0.1 IU/ml. The immunizing step isperformed passively or actively. The level of antibodies of tetanustoxin present in blood plasma is determined by antibody titer or anyapplicable other method known in the art.

In each of the embodiments of the foregoing methods the therapeuticallyeffective amount of tetanus toxin is delivered at the selected site byinjection, topical application, aerosol, or instillation into ducts orbody orifices. In certain preferred embodiments of the foregoing methodsthe therapeutically effective amount of tetanus toxin is delivered tothe target neurons encapsulated into liposomes or artificial vesicleswith bi-layer lipid membranes.

In certain preferred embodiments of each of the foregoing methods, thetherapeutically effective amount of tetanus toxin is suspended in apharmaceutically acceptable carrier.

In other preferred embodiments of each of the foregoing methods, thetetanus toxin is in the form of freeze-dried powder.

In each of the foregoing embodiments of each of the foregoing methodsthe target neurons include motor neurons, autonomic neurons, sensoryneurons or neurons of the central nervous system.

In certain embodiments of each of the foregoing methods, the activity ofthe target neurons is affected by inhibiting directly or indirectly therelease of neurotransmitters or neuropeptides.

In certain preferred embodiments of the invention wherein the neuralactivity of the target neurons is reversibly inhibited or the targetneurons are denervated, the therapeutically effective amount of TT isfrom about 100 units to about 10,000 units for the selected site.

In other more preferred embodiments wherein the neural activity of thetarget neurons is reversibly inhibited or the target neurons aredenervated by administration of tetanus toxin, the therapeuticallyeffective amount of TT is from about 500 units to about 5000 units forthe selected site.

In other most preferred embodiments of each of the foregoing methods ofreversibly inhibiting the neural activity of or denervating the targetneurons, the therapeutically effective amount of TT is from about 1000units to about 2000 units for the selected site.

In certain other preferred embodiments of each of the foregoing methods,wherein the administration of tetanus toxin results in the increase ofthe neural activity of the target neurons or in an excitatory responseof the nerve function at the selected site innervated by the targetneurons, the therapeutically effective amount of TT is from about 0.01units to about 2000 units for the selected site. In certain morepreferred embodiments of each of the foregoing methods, thetherapeutically effective amount is from about 1 unit to about 10 unitsfor the selected site. In certain most preferred embodiments of theforegoing methods, the therapeutically effective amount is from about 2units to about 4 units for the selected site.

In certain other embodiments of each of the foregoing methods, wheneverthe tetanus toxin is administered at a selected site it evokes anexcitatory response in target neurons associated with tissues or organsof the skeletal muscles. For these foregoing embodiments, applicableclinical disorders include without limitation sleep apnea and snoring,scoliosis, strabismus, muscle atrophy, neurologically impaired musclesincluding muscular dystrophy, ALS, or myasthenia gravis, decrease inmuscle mass, or decrease in facial muscle tone.

In other embodiments of each of the foregoing methods, whenever theadministration of TT evokes an excitatory response of neural activity insmooth muscles such as tissues or organs including without limitationlower esophageal sphincter, anal sphincter, bladder, bladder sphincter,vaginal sphincter, pyloric sphincter, upper esophageal sphincter, colonwall muscles.

In other embodiments of the invention, wherein the methods of theinvention include administration of TT evoking an excitatory response oftarget neurons of the autonomic, parasympathetic nervous system, theselected sites include tissues or organs affecting saliva production,the organs affecting saliva production including the submandibulargland, parotid gland, sublingual gland, or minor salivary glands of theoral mucosa.

In yet other embodiments, wherein the methods of invention includeadministration of TT to evoke an excitatory response of target neuronsof the autonomic sympathetic nervous system, the selected site comprisestissues or organs affected by nasal congestion, impotence, hair loss orhypotension.

In certain other embodiments of the foregoing methods, the tetanus toxinis administered to skeletal muscles to evoke a reversible inhibitoryresponse at the selected sites including tissues or organs affected byspastic dysphonia, hemifacial spasm and blepharospasm, temporalmandibular join syndrome or bruxism, torticollis, neck pain, writer'scramp, limb muscle contracture, nerve regeneration within a muscle ormigraine headache. The applicable skeletal muscles include vocal folds,facial muscles, masseter muscle, sternocleidomastoid muscle, trapeziusmuscle, forearm muscles, limb muscles, temporalis muscles and otherunspecified muscles.

In other embodiments of the foregoing methods, wherein theadministration of TT evokes a reversible inhibitory response, thetetanus toxin is administered to other selected sites including tissuesor organs of smooth muscles affected by bronchospasm, cricopharyngealspasm, esophageal spasm, achalasia, obesity, spastic colon or analfissures. The muscles include pulmonary smooth muscles, cricopharyngeusmuscle, esophagus, lower esophager sphincter, stomach wall muscles,colon wall muscles and anal sphincter.

In yet other embodiments of the foregoing methods, tetanus toxin evokesreversible inhibitory responses by administering it at the selected siteincluding tissues or organs affected by gastric acid, prostatehypertrophy, rhinorrhea, salivation, irritation of pulmonary mucosa,psoriasis, immune tolerance or immune reaction. In the foregoingembodiments, the applicable target neurons are part of the autonomicparasympathetic system and include gastric nerve supply, prostate gland,intranasal mucosa, pulmonary mucosa, submandibular gland, skin andthymus. In certain other embodiments of the foregoing methods, otherselected sites for TT application include tissues or organs affected byosteoporosis or angina including bones, coronary arteries and cardiacmuscles.

In certain other embodiments of each of the foregoing methods TT can beadministered with a vasoconstrictor at the target neurons of theselected site in an amount from about 1:200,000 to about 1:100,000. Thevasoconstrictor can be administered prior to, contemporaneously with orimmediately after the administration of the tetanus toxin.Vasoconstrictors useful in the present invention include withoutlimitation epinephrine, norepinephrine, or epinephryl borate.

In another aspect, the invention is directed to a pharmaceuticalformulation for modulating a neural function of an animal at a selectedsite affected by target neurons, the formulation comprising atherapeutically effective amount of tetanus toxin suspended in apharmaceutically acceptable carrier for delivery to the selected site,wherein the therapeutically effective amount of tetanus toxin is fromabout 100 units to 10,000 units for the selected site.

In another aspect, the invention is directed to a pharmaceuticalformulation for decreasing the activity of a nerve function in an animalat a selected site, the formulation comprising a therapeuticallyeffective amount of tetanus toxin suspended in a pharmaceuticallyacceptable carrier for delivery to the selected site, wherein thetherapeutically effective amount of tetanus toxin is from about 100units to 10,000 units for the selected site. In the foregoing embodimentthe selected site includes tissues or organs affected by spasticdysphonia, hemifacial spasm and blepharospasm, temporal mandibular joinsyndrome, bruxism, torticollis, neck pain, writer's cramp, limb musclecontracture, migraine headache, bronchospasm, cricopharyngeal spasm,esophageal spasm, achalasia, obesity, spastic colon, anal fissures,gastric acid, prostate hypertrophy, rhinorrhea, salivation, irritationof pulmonary mucosa, psoriasis, immune tolerance or immune reaction,osteoporosis or angina.

In yet another aspect, the invention relates to a pharmaceuticalformulation for increasing the activity of a nerve function in an animalat a selected site, the formulation comprising a therapeuticallyeffective amount of tetanus toxin suspended in a pharmaceuticallyacceptable carrier for delivery to the selected site wherein thetherapeutically effective amount of tetanus toxin is from about 0.001units to about 2000 units for the selected site. The selected site forthe pharmaceutical formulation of this embodiment comprises tissues ororgans affected by sleep apnea and snoring, scoliosis, strabismus,muscle atrophy, neurologically impaired muscles including musculardystrophy, ALS, myasthenia gravis, decrease in muscle mass, decrease infacial muscle tone, nasal congestion, impotence, hair loss orhypotension. Other selected sites for the pharmaceutical formulation ofthis embodiment include tissues or organs including lower esophagealsphincter, anal sphincter, bladder, vaginal sphincter, pyloricsphincter, upper esophageal sphincter, colon wall muscles. Otherselected sites for the pharmaceutical formulation of this embodimentinclude tissues or organs affecting saliva production or nasal mucosa,the organs affecting saliva production including submandibular gland,parotid gland, sublingual gland, or minor salivary glands of the oralmucosa.

The invention is further directed to the use of tetanus toxin in thepreparation of a medicament for a method of treating a clinical disorderor symptom of an animal, comprising administering a therapeuticallyeffective amount of tetanus toxin to a selected site affected by targetneurons related to the clinical disorder or symptom of the animal,wherein the therapeutically effective amount of tetanus toxin is fromabout 100 units to 10,000 units of the selected site.

Another aspect of the invention is directed to the use of tetanus toxinin the preparation of a medicament for a method of treating a clinicaldisorder or symptom in an animal, comprising administering atherapeutically effective amount of tetanus toxin at a selected site ofthe animal in order to decrease the activity of a nerve function in theanimal, the nerve function related to the clinical disorder or symptomof the animal, wherein the tetanus toxin causes an excitatory orreversible inhibitory response of the nerve function at the selectedsite innervated by the target neurons, wherein the therapeuticallyeffective amount of tetanus toxin is from about 100 units to 10,000units of the selected site. In this aspect of the invention, theselected site comprises tissues or organs affected by spastic dysphonia,hemifacial spasm and blepharospasm, temporal mandibular join syndrome,bruxism, torticollis, neck pain, writer's cramp, limb musclecontracture, nerve regeneration within a muscle, migraine headache,bronchospasm, cricopharyngeal spasm, esophageal spasm, achalasia,obesity, spastic colon, anal fissures, gastric acid, prostratehypertrophy, rhinorrhea, salivation, irritation of pulmonary mucosa,psoriasis, immune tolerance or immune reaction, osteoporosis or angina.

In yet another aspect, the present invention is directed to the use oftetanus toxin in the preparation of a medicament for a method oftreating a clinical disorder or symptom in an animal, comprisingadministering a therapeutically effective amount of tetanus toxin at aselected site of the animal in order to increase the activity of a nervefunction in the animal, the nerve function related to the clinicaldisorder or symptom of the animal, wherein the tetanus toxin causes anexcitatory or reversible inhibitory response of the nerve function atthe selected site innervated by the target neurons, wherein thetherapeutically effective amount of tetanus toxin is from about 0.001units to about 2000 units for the selected site. In this embodiment ofthe invention the selected site comprises tissues or organs affected bysleep apnea and snoring, scoliosis, strabismus, muscle atrophy,neurologically impaired muscles including muscular dystrophy, ALS,myasthenia gravis, decrease in muscle mass, decrease in facial muscletone, nasal congestion, impotence, hair loss or hypotension. Otherselected sites for the foregoing embodiment include tissues or organsincluding lower esophageal sphincter, anal sphincter, bladder, bladdersphincter, vaginal sphincter, pyloric sphincter, upper esophagealsphincter, colon wall muscles. Other selected sites for the foregoingembodiment comprise tissues or organs affecting saliva production ornasal mucosa, the organs affecting saliva production includingsubmandibular gland, parotid gland, sublingual gland, or minor salivaryglands of oral mucosa.

Yet another aspect to the invention is directed to a method ofmodulating a cellular, non-neural activity of an animal at a selectedsite, the method comprising administering at the selected site atherapeutically effective amount of the tetanus toxin, wherein thecellular activity includes release of a cellular component comprisinghormones, inflammatory modulators from nerves or blood cells,cholinergic caused secretions, mucus secretions from respiratory,digestive or urinary glands.

In the foregoing embodiment, the cellular activity occurs in cellsincluding macrophages, monocytes, endocrine cells or renal cells. Inthis embodiment the cellular activity is modulated over a period fromabout one hour to about one year. In a preferred embodiment the cellularactivity can be controlled or modulated over a period of one week tofour months.

In certain embodiments of each of the foregoing methods related tomodulating cellular activity, each of the foregoing methods furthercomprises: (i) determining the level of antibodies of tetanus toxinpresent in blood plasma of the animal prior to administering of anytetanus toxin; and (ii) immunizing the animal when the level of tetanustoxin is below 0.1 IU/ml. The immunizing is performed passively oractively. The level of antibodies present in blood plasma is determinedby antibody titer.

In certain embodiments of each of the foregoing methods thetherapeutically effective amount of tetanus toxin is delivered at theselected site by injection, topical application, aerosol, instillationinto ducts or body orifices, encapsulated into liposomes or artificialvesicles with bi-layer lipid membrane. In the foregoing embodiments thetherapeutically effective amount of tetanus toxin is suspended in apharmaceutically acceptable carrier. Additionally, the tetanus toxin canbe in the form of a freeze-dried powder.

In the embodiments of the foregoing methods related to modulatingcellular activity the therapeutically effective amount of tetanus toxinis from about 0.001 units to about 10,000 units for the selected site.

In other more preferred embodiments of the foregoing methods, thetherapeutically effective amount of tetanus toxin is from about 1 unitto about 5000 units for the selected site.

In other most preferred embodiments of the foregoing methods, thetherapeutically effective amount of tetanus toxin is from about 10 unitsto about 1000 units for the selected site.

In certain embodiments of each of the foregoing methods the selectedsite comprises tissues or organs affected by malignant carcinoma orinflammatory conditions.

In yet another aspect, the invention relates to a pharmaceuticalformulation for modulating a cellular activity of cells of an animal,the formulation comprising a therapeutically effective amount of tetanustoxin suspended in a pharmaceutically acceptable carrier for delivery tothe selected site, wherein the therapeutically effective amount oftetanus toxin is from about 0.001 units to about 10,000 units for theselected site.

In another more preferred embodiment, the invention relates to thepharmaceutical formulations of the foregoing methods, wherein thetherapeutically effective amount of tetanus toxin is from about 1 unitto about 5000 units for the selected site.

In yet another most preferred embodiment, the invention related to thepharmaceutical formulations of the foregoing methods, wherein thetherapeutically effective amount of tetanus toxin is from about 10 unitsto about 1000 units for the selected site.

In certain embodiments of the invention the pharmaceutical formulationsare administered at a selected site comprising tissues or organsaffected by malignant carcinoma or inflammatory conditions.

Another aspect of the invention is directed to the use of the tetanustoxin in the preparation of a medicament for a method of effectivelytreating a clinical disorder or symptom in an animal, comprisingadministering a therapeutically effective amount of tetanus toxin inorder to modulate a cellular activity of an animal at a selected site,the cellular activity including release of a cellular componentincluding hormones, inflammatory modulators from nerves or blood cells,cholinergic caused secretions, mucus secretions from respiratory,digestive or urinary glands, wherein the therapeutically effectiveamount of tetanus toxin is from about 0.001 units to about 10,000 unitsfor the selected site.

In a preferred embodiment the invention is directed to the use of thetetanus toxin in the preparation of a medicament for a method ofeffectively treating a clinical disorder or symptom in an animal,comprising administering a therapeutically effective amount of tetanustoxin in order to modulate a cellular activity of an animal at aselected site, the cellular activity including release of a cellularcomponent including hormones, inflammatory modulators from nerves orblood cells, cholinergic caused secretions, acid secretions mucussecretions from respiratory, digestive or urinary glands, wherein thetherapeutically effective amount of tetanus toxin is from about 1 unitto about 5,000 units for the selected site. In a most preferredembodiment the use of the tetanus toxin is in a therapeuticallyeffective amount from about 10 units to about 1000 units for theselected site. In the foregoing uses of tetanus toxin, the selected sitecomprises tissues or organs affected by malignant carcinoma orinflammatory conditions.

The invention is also directed to a method for the alleviation of painexperienced by an animal comprising administering tetanus toxinsuspended in a pharmaceutically acceptable carrier to a selected site ofthe animal, the tetanus toxin in a therapeutically effective amountsufficient to decrease or reversibly inhibit the release of inflammatoryneurotransmitters or neuropeptides associated with the pain.

In another aspect, the invention relates to a method for the alleviationor blocking of pain sensation experienced by an animal comprisingadministering tetanus toxin to a selected site of the animal in atherapeutically effective amount sufficient to denervate sensory neuronsaffecting the release of inflammatory neurotransmitters or neuropeptidescontrolling the selected site of the animal.

In yet another aspect, the invention is directed to a method for theincrease of muscle mass in an animal comprising administering to aselected site of a muscle a pharmaceutically effective amount of tetanustoxin sufficient to cause an increase or an excitatory response in theneural activity of the selected muscle. In the foregoing method theselected muscle is a skeletal or smooth muscle.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples which are set forth herein.The detailed description and the examples enhance the understanding ofthe invention, but are not intended to limit the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Methods and pharmaceutical compositions for modulating a neural functionof an animal by the administration of an effective amount of tetanustoxin to an animal are provided. These methods and compositions can beused for management or treatment of many clinical disorders or diseases.

As used herein, in the context of modulating or controlling a neuralfunction, a “selected site” is defined to include a tissue or organaffected directly or indirectly by a target neuron. In the context ofmodulating non-neural, cellular activity, a “selected site” refers to atissue or organ affected directly or indirectly by a non-neural cellularby including release of a cellular component such as hormones,inflammatory modulators from nerves or blood cells, cholinergic causedsecretions, acid secretions, mucus secretions from respiratory,digestive or urinary glands.

As used herein, the term “modulating” is used interchangeably with theterm “controlling” and means to adjust to a requirement or regulate.

As used herein, the term “target neurons” refers to a neuron that isaffected directly or indirectly by the presence of tetanus toxin in atherapeutically effective amount sufficient to cause a physiologicalchange at the selected site. Target neurons include without limitationneurons functionally clarified as sensory neurons, motor neurons,interneurons, autonomic and central nervous system neurons particularlyrelated to uptake, transport or inhibition of neurotransmitters orneuropeptides.

The term “therapeutically effective amount” means that the tetanus toxinis administered in a non-toxic amount sufficient to cause reduction inthe occurrence or magnitude of the symptoms being targeted. When thetoxin is used to increase the neuronal tone, a preferred amount may beequal to the amount of botulinum toxin that causes the opposite effect(denervation). When tetanus toxin is used to cause denervation of thetarget neurons, the amount may be about 500 times greater then the sameamount of botulinum toxin required to achieve a similar effect.

As used herein, the term “patient” broadly refers to any animal that isto be treated with the compositions and by the methods herein disclosed.In particular, the disclosed methods and compositions will find use inveterinary practice and animal husbandry for, e.g., birds and mammal,wherever modulation of neural function is convenient or desirable. Theterm “animal” includes all members of the animal kingdom, includingmammals and especially humans.

For purposes of the present invention, tetanus toxin is commerciallyavailable from for example, Lederle Laboratories of Wayne, N.J. underthe tradename “Tetanus Toxoid Purogenated.” The method of the inventionwill preferably encompass the use of pharmaceutically safe forms of theintact tetanus toxin, including both heavy and light chains, as well asany fragment thereof, such as an AB fragment. Combinations with othermoieties such as other proteins including a hybrid of a protein and theheavy or light chain portion of the tetanus toxin are also included inthe present invention.

More specifically, for purposes of the present invention, the use oftetanus toxin, as well as a fragment thereof, is contemplated. Inaddition, it may be beneficial to separate the binding protein portionof the tetanus toxin to use in association with other proteins to allowthe other proteins to enter a cell. It may also be beneficial to bindthe toxic protein portion of the tetanus toxin to other proteins toallow the toxic protein to enter cells it would otherwise be incapableof entering.

Those of ordinary skill in the art will know, or can readily ascertain,how to obtain tetanus toxins, in a pharmaceutically safe form,preferably, a form that is nonteratogenic. For most of the neurotoxinsof the invention, pharmaceutical safety will be dose-dependent such thatrelatively low dosages of toxin will be “safe” as compared to dosageswhich are known to be sufficient to produce disease.

Preferably, the tetanus toxins of the invention will be administered asa composition in a pharmaceutically acceptable carrier. To that end,presynaptic neurotoxin compositions are prepared for administration bymixing a toxin of the desired degree of purity with physiologicallyacceptable sterile carriers. Such carriers will be nontoxic torecipients at the dosages and concentrations employed. Ordinarily, thepreparation of such compositions entails combining the neurotoxin withbuffers, antioxidants such as ascorbic acid low molecular weight (lessthan about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose or dextrins, chelating agents such asEDTA, glutathione and other stabilizers and excipients. Suchcompositions may also be lyophilized and will be pharmaceuticallyacceptable; i.e., suitably prepared and approved for use in the desiredapplication.

Unlike other pharmaceuticals, the biological activity of the same weightof a neurotoxin commonly varies between the different laboratories thatproduce the toxin. The reason for this is that not all preparationscompletely remove other proteins or that during isolation of the toxinsome molecules tend to lose their biological activity.

Measurement by a biological assay is normally used to obtain a standardpreparation of the tetanus toxin. A common biological assay used tomeasure the toxicity of tetanus toxin is the average dose needed to kill50% of mice when injected subcutaneously. This amount of toxin isreferred to as the mouse LD₅₀, or the mMLD (mouse minimum lethal dose),or simply as a unit of tetanus toxin.

Another biological assay commonly used in experimental studies ofneurotoxins is the mouse hemi diaphragm preparation (Bulbring, E., JPhys (London), 1, 38-61, (1946)). This is an in vitro assay consistingof half of the diaphragm of a mouse and the nerve that innervates it.The nerve is electrically stimulated to cause the diaphragm to contractand the effect of different toxin preparations on blocking theneuromuscular transmission can be precisely measured. The advantage ofthis particular assay is that it precisely measures the effect of toxinat the neuromuscular junction, which is the neural connection that isusually of interest to investigators as well as clinicians.

Except for vaccine preparations, tetanus toxin is not used commercially;there is no current standard that composes a unit. Instead, the unit hasbeen reported by Haberman, et al. to vary from 0.3 ng/kg to 25 ng/kg(Habermann, et al., Naunyn Schmiedebergs Arch Pharmacol, 311(1), 3340,(1980)). The dose of tetanus toxin needed for a local effect will varydepending on the desire for excitatory or inhibitory action, thespecific type of neural tissue (Montecucco, C. and G. Schiavo, Q RevBiophys, 28(4), 423-72 (1995)), the size of the tissue or organ in whichthe effect is desired, the species to be injected, the activity of thenerve, temperature (Habermann, E., F. Dreyer, and H. Bigalke, NaunynSchmiedebergs Arch Pharmacol, 311(1), 3340, (1980)) and other factors.

There is no absolute standard weight that constitutes a unit of tetanustoxin, and as tetanus toxin has never been used for clinicalapplications there has never been any compelling reason to adopt astandard. In future clinical applications of tetanus toxin a commercialvendor would be required to produce and distribute a standardized andconsistent preparation of tetanus toxin in unit dose form. At presentthere is no standard, and the weight of tetanus toxin constituting aunit that is currently available from different companies orlaboratories varies from approximately 0.1 ng to 100 ng, with most beingapproximately 1 ng. For this reason, as used in the present invention aunit of tetanus toxin means 0.02 ng of tetanus toxin.

In contrast to BT, TT has never been used for clinical applications.This is surprising as TT has a variety of special qualities that makesit potentially much more clinically useful then BT. First, it binds toall classes of neurons: motor, autonomic, and sensory. In contrast thebotulinum toxin binds to motor neurons. Secondly, TT is transported tothe central nervous system. Although the toxin binds to peripheralnerves, mostly motor neurons, it does not cause its primary effect byintoxicating the peripheral neuron. Instead the toxin is transportedwithin the axons of the motor neurons to their cell bodies located inthe spinal column and brain stem. There the motor neurons release thetoxin, allowing to pass the synapse and bind and enter into neurons ofthe central nervous system. Thirdly, it blocks the release of most ifnot all neurotransmitters: acetylcholine, norepinephrine, epinephrine,dopamine, glutamate, GABA, glycine, serotonin, as well as neuropeptidesCGRP, neuropeptide Y, substance P and others; and neuroendocrinehormones oxytocin, vasopressin. In the central nervous system the toxinbinds preferentially to neurons that use the neurotransmitters GABA andglycine. After binding the toxin enters into these neurons and blocksthe release of these neurotransmitters. The GABA and glycine containingneurons are the inhibitory neurons of the central nervous system.Normally a motor neuron receives input from many inhibitory andexcitatory neurons and these opposing influences largely cancel eachother. However after inhibitory neurons are blocked the excitatoryneurons can stimulate the motor neurons unopposed so that the motorneuron activity increases causing increased activity in the muscleinnervated by these intoxicated motor neurons. Clinically this is seenas increased muscle tone that reaches an unremitting spasm in its finalstages. Fourthly, TT can cause increased activity of peripheral andcentral nerves as well as blocking these same nerves depending on theexact dosage used.

The mechanism of action of tetanus toxin is such that the excitatoryaction evoked by its use is indirect. The tetanus toxin is taken up by aperipheral neuron such as the motor neuron innervating a muscle and istransported back to the cell body of the motor neuron in the spinalcolumn. It is then released into the presynaptic space. Motor neuronsare connected to many other neurons that are either excitatory orinhibitory. Under normal circumstances these inputs are balanced so thatthe motor neuron is excited just enough to perform the appropriatemuscle movements. The inhibitory neurons use the neurotransmittersglycine or GABA, and the tetanus toxin binds to and then blocks theseneurons (Bigalke, H., et al., Naunyn Schmiedebergs Arch. Pharmacol,316(2), 143-8, (1981)). When the inhibitory neurons are blocked only theexcitatory input remains and the activity of the muscle increases.

Each muscle has a different mixture of excitation and inhibition. Forexample the masseter muscle is a muscle that has a great deal ofexcitatory input. If tetanus toxin blocks all the inhibitory input tothe motor neurons supplying the masseter muscle it goes into anextremely strong and prolonged spasm. This spasm of the masseter muscleclenches the jaw shut and is the origin of the term “lockjaw” thatrefers to the systemic disease.

In most muscle applications it is not desirable to inject the tetanustoxin to obtain a prolonged spasm. Instead, what is usually needed is amild to moderate increase in muscle tone. Therefore, very low doses areused for local excitatory applications. For example, when the hind limbof a cat was injected with 1.5 ng into the triceps surae muscle of thehind leg, the tetanus toxin caused an incomplete extension of the legdeveloped after about one week (Takano, et al., Toxicon 27(4), 431-8,(1989)). When 7.5 ng or more were injected the leg of the cat remainedextended in a stiff continuous spasm that the animal could not overcome.

Similarly, excitatory applications in human muscle or other targettissue requires doses in the range of 0.001 to 10 ng depending on thesize of the muscle and its underlying neural mixture of excitatory andinhibitory inputs. To achieve the desired application, those skilled inthe art understand that it is useful to begin by using a dose at thelower end of the foregoing range and wait to observe the effects.Subsequent increasing doses of tetanus toxin can be given until theproper level is achieved.

Tetanus toxin can also block peripheral neural transmission directly atthe neuromuscular junction to cause a flaccid paralysis. Experimentsshow that this direct neural blocking effect requires approximately 500times more tetanus toxin than those used to cause excitation (Habermann,E., et al., Naunyn Schmiedebergs Arch Pharmacol, 311(1), 33-40, (1980)).The reason for this large disparity in dosages causing the differenteffects of excitation or inhibition is unknown.

As local paralysis may require 0.5 to 50 ng of tetanus toxin or more toachieve its effect, these doses approach or exceed the lethal dose for anon-immunized human. The lethal dose for a non-immunized human isapproximately 2.5 ng/kg or 175 ng for a 70 kg individual. Therefore useof tetanus toxin for neural block may be restricted to smaller targetsrequiring low amounts of toxin. It would, therefore, be critical toascertain the immune status of the human be known prior to administeringthe tetanus toxin. Universal immunization is performed in the UnitedStates and most industrialized nations. Immune status is measured ininternational units of tetanus antibody. A blood plasma antibody contentgreater than 0.1 IU/ml is considered protective against systemictetanus. The level of immune status is high after immunization andgradually falls over in the following years. Studies have shown thathalf of vaccinated human adults have less then 0.11 IU/ml. Suchindividuals would probably require booster vaccination to raise theirlevels of immunity.

Tetanus toxin can also block action potentials in nerve axons just likelocal anesthetics. As a result, tetanus toxin can be used to inject andblock any nerve along its course, thus increasing its clinicalusefulness. Unlike botulinum toxin, tetanus toxin binds to the membraneof the neuronal axon (Herreros, et al., European Journal Neurosci 9(12),2677-86, (1997)). Experimental animal models of local tetanus show largeamounts of the toxin along the course of the nerve leading from theinjected muscle back to the spinal column outside of the axons (Erdmann,et al., Neuryn Schmiedebergs, Archive of Pharmacology, 290(4), 357-373,(1975)). Physiological studies of patients with local tetanus suggestthat the nerve conduction is decreased or blocked, which is a separateeffect from the block at synapses, (Dastur, F. D., et al., Journal OfNeurology, Neurosurgery And Psychiatry 40(8), 782-6 (1977)).

Also many of the proposed injections might require the use ofelectromyography for proper localization within the muscle. This usesthe tip of the needle to sense muscle activity and is routine in manybotulinum toxin injections.

Unexpectedly large injections of tetanus toxin can be made into a musclewithout systemic or regional spread. Partly this is due to extremelyhigh affinity that the tetanus toxin has for the neural membrane thatcauses it to bind rapidly with neurons (Critchley, D. R., et al. J CellBiol, 100(5), 1499-507 (1985)). Additional mechanisms can be used toreduce the risk of side effects. An example would be to add adrenaline1:100,000 dilution, phenylephrine ½% Or other vasoconstricting agents tothe injection. These would cause a temporary local vasoconstriction anddecrease in blood flow, thereby decreasing the opportunity for thetetanus toxin to enter the systemic circulation.

Other vasoconstrictors useful in the present invention include withoutlimitation epinephrine, norepinephrine, or epinephryl borate.

Other additional precautions can be taken to prevent the systemic spreadof tetanus toxin when large doses are injected locally. For example, thetetanus anti toxin can be injected, either into the same site as tetanustoxin injection but with some time delay or at a distant site (Fezza, J.P., et al., Opthalmic Plastic and Reconstructive Surgery, 16(2),101-113, (2000)). In the rabbit the lethal dose of tetanus toxin in atypical 2 kg rabbit is approximately 1-10 ng or 0.5-5 ng/kg. However, aninjection of 25 ng, 2,500 to 25,000 times the lethal dose, was injectedsafely into the orbicularis oculi muscle. In this experiment 250 IU oftetanus antitoxin were simultaneously injected intramuscularly into ahind limb muscle and blocked any systemic spread. At five days theseanimals demonstrated paresis of the injected eyelid without any local orsystemic spread of toxicity. Clearly this is at the highest end ofdosage, spectrum as either reducing the amount of tetanus toxin to 125IU, or increasing the injected dose of tetanus toxin to 37.5 ng,resulted in local or systemic signs of toxicity (Fezza, J. P., et al.,Opthalmic Plastic and Reconstructive Surgery, 16(2), 101-113, (2000)).

In certain preferred embodiments, the tetanus toxin is used to controlmotor neuron function. For example, the toxin is administered locally toa particular target site in the body (e.g., particular muscles) in asufficient amount to increase the neural activity of the motor neuronsin the target area. This in turn increases neural stimulation of musclecells innervated by said neurons. This results in increased muscle tone,and if the muscle is immobilized ill a shortened length, it will rapidlyadapt to the shortened length (Abe, Y, et al., Acta Otolaryngol(Stockh), 112 (4), 703-9, (1992)). Alternatively, by adjusting theamount of the tetanus toxin, one may produce an opposite effect, e.g.,denervation of the neurons. For a clinically beneficial effect, atherapeutically effective amount of the tetanus toxin is administered.

In preferred embodiments, low concentrations of tetanus toxin toincrease tone are administered to genioglossus, geniohyoid and softpalate muscles (e.g., for treatment of sleep apnea); pharyngeal muscles,to aid swallowing in patients with dysphagia; paraspinal muscle (e.g.,for treatment of scoliosis); extraocular muscle (e.g., for treatment ofstrabismus); muscles in the immobilized limb (e.g., to prevent atrophyduring long-ten-a casting); to different muscle in paralyticneurological diseases such as ALS to restore muscle tone; loweresophageal sphincter (e.g., to control esophageal reflux); stomachmuscle (e.g., for gastric contracture and decreased appetite); facialmuscles (e.g., for increased tone and youthful appearance); targetmuscles to increase muscle mass as a substitute for exercise.

In other preferred embodiments high concentrations of tetanus toxin todecrease tone are administered to facial muscles to decrease facialmuscle or eyelid spasm; to temporal muscles to treat myofascial pain andheadache; to cervical muscles, to treat torticollis and cervicaldystonia; various target muscles for muscular dystrophy, ALS, myastheniagravis; limb muscles (e.g., to treat spasm or contracture resulting fromupper motor neuron lesions, such as seen after strokes); to jaw musclesto decrease bruxism; to laryngeal muscles to treat spasmodic dysphoniaand hyperfunctional conditions; to forearm muscles to treat writerscramp; to leg muscles to treat night cramps; in any muscle whereincreased branching of motor nerves is beneficial such as during nerveregeneration after traumatic nerve injury.

In certain embodiments, the tetanus toxin may be administered locally toa particular part of the autonomic system (e.g., target tissue or organ)to control the activity of the neurons in that area, which also in turnaffects the target autonomic system innervated by said neurons.

In certain embodiments, the tetanus toxin is administered locally to atarget autonomic system (e.g., tissue or gland) in a therapeuticallybeneficial low amount sufficient to increase the activity of the neuronsin that area. This results in increased stimulation of the cells (e.g.,of tissue or gland) innervated by said neurons. Tetanus toxin may beadministered to salivary, lacrimal and vaginal glands to treat drymouth, dry eye and atrophic vaginitis; to mammary glands to increasemilk production; to nasal mucosa to treat nasal congestion and allergicsymptoms; to penile vasculature tissue to prolong erections and treatimpotence; to pancreas and other endocrine glands to increase hormoneproduction; to colon and other gastrointestinal organs to increasemotility to treat constipation; to sympathetic nerves of the lung torelax smooth muscle in asthma and chronic obstructive diseases; togastric smooth muscle to cause gastric shrinkage and cause feelings ofsatiety to decrease appetite and cause weight loss; to pulmonary mucusglands to increase serous mucous production and cilia transport to treatcystic fibrosis; to adipose tissue to cause lipolysis and fat cellshrinkage.

In certain other embodiments, higher doses may be used to decreaseautonomic neural activity. When used clinically, the tetanus toxin isadministered in a therapeutically effective amount to hair follicles totreat hair loss; to prostate glands to cause shrinkage of an enlargedprostate; to connective tissue to increase its metabolism to treat thelax skin of the aged; to pain fibers to decrease pain sensation andinflammation; to skin in proliferative or allergic diseases such aspsoriasis and atopic dermatitis; to sebaceous glands of skin to treatacne; to sebaceous glands of ear canal skin to decrease ear wax; tosympathetic nerves of the circulatory system to decrease blood pressure;to neuromodulate the immune response in the thymus and spleen, lymphnodes, or any tissue where neural immune interactions exist; to skin,digestive tract or mucosa to prevent recognition of foreign antigens toproduce tolerance; to tonsils to decrease their size; to the anteriorchamber of the eye to decrease fluid production to treat ocularhypertension; to gastric mucosa to decrease acid production in refluxesophagi is; to the nasal mucosa to decrease rhinorrhea, and to decreasethe neural influence on mast cell histamine release to decrease allergicsymptoms; to pterygopalatine ganglia to block vasodilatory neurons toprevent true migraine headache.

In certain preferred embodiments, the tetanus toxin is administered tothe target autonomic system in a therapeutically effective amount totreat nasal congestion rhinorrhea and allergic symptoms (Ado, A. D.,Eksperimentalnaia I Klinicheskaia Farmakologiia, 58 (3), 43-5 (1995);Albegger, K., Hno, 36 (10), 389-98 (1988); Agro, A. et al., Advances InNeuroimmunology, 5 (3), 311-9, (1995)) modulate immune responses (Ado,A-D., Vestnik Rossiiskoi Akademii Meditsinskikh Nauk, (7), 48-51 (1993);Albanese, A., et al., Mov Disord, 12(5), 764-6, (1997)), relax analsphincters in constipation (Sabbadini, E. et al.,Neuroimmunonmodulation, 2 (4), 184-202, (1995)) affect penile erection,decrease inflammation and pain in various organs, decrease skinproliferation in diseases such as psoriasis, invoke antigen tolerance,decrease blood pressure, decrease migraine headache, increase ordecrease salivation, decrease sweating, decrease the size of theprostate gland, increase the connective tissues, and control hair loss.

In certain other embodiments, the tetanus toxin is administered tosensory neurons to cause a reversible sensory block. One application ofsuch use would be to block pain from any part of the body, e.g., bylocally administering to that part an amount effective to block ordecrease the pain. Another embodiment would be to block the inflammatorymediators released by sensory neurons, e.g., by locally administering toa joint a therapeutic beneficial amount in rheumatoid arthritis.

In certain other embodiments tetanus toxin is applied to parts of thecentral nervous system, either directly or as a result of retrogradetransport from a peripheral nerve.

In certain other embodiments tetanus toxin is administered tonon-neuronal cells for beneficial effect. These include macrophages andother white blood cells to decrease inflammation, endocrine cells todecrease the secretion of hormones, parietal cells of the stomach todecrease acid production, fluid producing cells in the eye to decreaseintraocular pressure in glaucoma, malignant cells to decrease motilityand metastases.

The present invention is also directed to veterinary uses of tetanustoxin, e.g., to increase the muscle mass of a target veterinary animal.This includes milk and meat production.

According to the present invention, tetanus toxin may be administered byvariety of modes of administration. When administered locally, the modeof administration includes but is not limited to injection (includingpressure jet injectors), aerosolized (for nasal, upper airway or lungadministration), topical application (on skin and mucous membranes andon internal body surfaces (such as during surgery or in the treatment oftrauma) open wounds, and by instillation into ducts (salivary, mammary,lacrimal) or body orifices (urethra, anus, oral).

When administered locally to a particular target site, the tetanus toxinaffects the activity of the neurons in that area, preferably withouthaving a systemic effect. As tetanus toxin is taken up by axons it canbe administered along the course of a nerve to block or increase theneural activity received by a distant organ or tissue innervated by thenerve. In preferred embodiments, the tetanus toxin is administeredlocally to a target site in the body. However, in certain cases, localapplication can deliberately result in a wide distribution of the toxin.For example, the local application can be to the cerebrospinal fluid, sothat it is distributed to large parts of the central nervous system; orinto an artery to be distributed to the body part that the arteryperfuses. In certain embodiments, application of the tetanus toxin maybe systemic, e.g., into the systemic circulation so that there isdistribution throughout the body.

Local application of tetanus toxin at pharmacological levels causestheir uptake by local nerve endings and their retrograde transport tothe central nervous system (CNS). In local application encompassing allmeans of delivery, including but not limited to injection, e.g.,pressure jet injectors, and topical application. In the CNS the tetanustoxin is transported transynaptically and binds to inhibitory neurons.The result is the disinhibition of the peripheral neuron and an increasein its activity. The exact amount of the increase and its pattern isrelated to the biology of that particular neuron. For widespreaddistribution in the CNS such as the veterinary applications the toxincan be directly injected into the cerebrospinal fluid.

When the toxin is to be administered to cells that lack the necessarymembrane receptors, the toxin may be encapsulated into liposomes,artificial vesicles with bi-layer lipid membranes. The vesicles wouldmerge with cells in the area of injection and deliver the toxininternally. To increase specificity the surface of the liposomes can becoated with specific proteins such as antibodies or glycoproteins thatallow specific docking of the liposome to the target cell.

EXAMPLES

The invention is further described in the following examples. Theexamples are illustrative of some of the products and methods of makingthe same falling with in the scope of the present invention. They are,of course, not to be considered in any way restrictive of the scope ofthe invention. Numerous changes and modification can be made withrespect to the invention. The materials used in the examples hereinbeloware readily commercially available.

Excitatory intramuscular applications of tetanus toxin are illustratedin examples 1-18. Inhibitory responses elicited by application oftetanus toxin are illustrated in examples 19 to 37. Increased tone inthe nerves of the autonomic system is desirable in many conditions. Themechanism of action is similar to that observed in muscles. Retrogradetransport from the site of injection causes block of inhibitory afferentinput. Examples 30, 33 and 38 illustrate the use of tetanus toxin tomodulate or control cellular activity of holocrine secretions endocrinecells and macrophages.

Example 1 Sleep Apnea and Snoring

In this example, a 60 year old male with snoring and obstructive sleepapnea is injected with tetanus toxin by passing a needle through themucosa in the floor of the mouth. 1 unit of tetanus toxin is injectedinto both genioglossus muscles. The needle is advanced further until thegeniohyoid muscle is entered and a further 1 unit is injected into eachmuscle. The needle is removed and reinserted through the oral mucosa ofthe soft palate within 2 centimeters of the edge of the hard palate. 1unit of tetanus toxin is injected into each levator muscle. Within oneweek the incidence of snoring and obstruction during sleep decreases.

Sleep apnea and snoring are clinical conditions affecting genioglossusand/or geniohyoid, tensor and levator veli palatini muscles. Sleep apneais a common disorder in which soft tissue of the upper airway (tongueand soft palate) impede the flow of air during inspiration therebycausing a partial obstruction to airflow and vibration of the softtissue of the area (snoring) or complete obstruction to airflow. Theresult of the obstruction includes disturbed sleep patterns, snoring,daytime somnolence, difficulty in concentrating, and contributes to mooddepression, hypertension and cardiac disease. The pathophysiology ofobstructive sleep apnea includes a decrease in activity of thegenioglossus and other upper airway muscles. The genioglossus muscleinserts into the base of the tongue and has phasic activity synchronouswith inspiration that moves the tongue forward to dilate the airway. Thegeniohyoid inserts into the hyoid bone and has a similar inspiratoryactivity. The tensor and levator veli palatini also have inspiratoryactivity that moves the soft palate superiorly. In this embodiment,administration (e.g., injections) of 1 unit of tetanus toxin into thegenioglossus and/or geniohyoid, tensor and levator veli palatini musclescan result in increased amplitude of the phasic motions and decrease theairway obstruction.

Example 2 Scoliosis Paraspinal Muscle

A female patient age 10 suffering from scoliosis, curvature of thespine, is treated by injection of 100 units of tetanus toxin intoparaspinal muscles. In 1-3 days the patient shows increased tone inmuscles that serve to straighten the spine.

The developmental misalignment of the spine that occurs with scoliosiscould be corrected with administration (e.g., injections) of tetanustoxin into the proper muscles that would straighten the spine. Thisremodeling of a bone by long-term increase in muscular activity hasnumerous other applications. Other examples include obtaining anexcitatory response from craniofacial muscles in order to rearrange thefacial skeleton.

Example 3 Strabismus

A male patient age 5, suffering from strabismus, or improper alignmentof the eyes, is treated by injection of 0.1 unit of tetanus toxin intothe medial rectus muscle of the misaligned eye. In 1 to 3 days the eyemoves into alignment. This example illustrates concept similar to thatdescribed in Example 2 except that rearrangement occurs in the muscularsoft tissue. Administration (e.g., injections) of tetanus toxin into thelateral rectus or other appropriate muscle increases its tonic activityand causes a straightening of the alignment of the globe.

Example 4 Preventing Muscle Atrophy

A male patient age 25 is suffering from a fracture of the femur and isscheduled to have a leg cast placed for 6 weeks. 10 units of tetanustoxin is injected into each muscle of the thigh. After 1 to 3 days thetone of the immobilized muscles increases. After 6 weeks the cast isremoved and the muscles show less atrophy then expected.

After a severe bone fracture or ligament tear, casting andimmobilization can result in atrophy of the muscles of the immobilizedlimbs. This undesirable side effect is prevented by the administration(e.g., via injection) of tetanus toxin into the muscles prior to castingto increase their tone and prevent atrophy.

Example 5 Esophageal Reflux Lower Esophageal Sphincter

In the lower esophageal sphincter, a 50 year old male with afflictedwith reflux esophagitis is injected with 1 unit of tetanus toxin. In 3days the symptoms of reflux acidity decrease.

Laxity in the lower esophageal sphincter results in reflux of acidcontents up the esophagus. This common medical problem can be preventedby administration (e.g., injection) of tetanus toxin into the loweresophageal sphincter. This example demonstrates that the increase oftone prevents esophageal reflux from occurring.

Example 6 Bladder or Bowel Incontinence Sphincters

A 50 year old female having urinary incontinence is injected with 1 unitof tetanus toxin into the external (pudendal nerves) and internal(sympathetic axons from the inferior mesenteric ganglion) urethralsphincters. In 3 days increased muscle tone in these sphincters relievesthe urinary incontinence.

In a related example, a 50 year old male with urinary incontinence isinjected with 1 unit of tetanus toxin into the urinary sphincter underdirect vision using a cystoscope. In 1-3 days the symptoms of urinarycontinence improve.

Many medical conditions result in incontinence, the inability to containurine or bowel contents. In those cases where decreased tone of thesphincter is the problem, administration (e.g., injections) of thetetanus toxin into the sphincter can increase tone. Additionalsphincters that could be injected include the vaginal introitus, pyloricsphincter, and upper esophageal sphincter.

Example 7 Gastric Contracture and Decreased Appetite

A 40 year old female suffering from obesity is injected with 100 unitsof tetanus toxin into the smooth muscle of the stomach wall and/orpyloric sphincter under direct vision using an endoscope. After 1 to 3days she feels more satiated and her appetite decreases.

It is known that the feeling of satiety is at least partly due todistension of the stomach. Some surgical procedures have been designedto take advantage of the effect by surgically decreasing the size of thestomach. Instead, administration (e.g., injections) of tetanus toxin canbe made into the stomach wall. The increased tone induced in theinnervation of the stomach smooth muscle would, over time, decrease itssize. The effect is a feeling of satiety after smaller amounts of foodintake.

Example 8 Muscular Dystrophy, ALS, Myasthenia Gravis

Many neurological disorders and aging are associated with a decrease inthe efferent activity to muscles. So long as a minimum number of motoraxons are still present, this activity can be increased withadministration (e.g., injections) of tetanus toxin. The particularmuscle is dependent on the conditions of the disease but includes allskeletal muscles.

Example 9 Muscle Contracture

A 60 year old female is suffering from spastic contraction of her rightarm after a cerebral vascular accident is injected with 10 units oftetanus toxin into the triceps muscle of the right arm. After 1-3 daysthe symptoms of contracture decrease and the arm rests in a moreextended position.

Contracture, stroke and other upper motor neuron lesions result in adisinhibition of limb muscles and especially the flexors of the upperlimb and the extensors of the lower. Administration (e.g., injections)of tetanus toxin can increase tone in the muscle opposite to thecontracture and result in a more neutral position of the limb.

Example 10 Facial Muscle Tone

Each orbicularis oculi muscle of a 70 year old female patient isinjected with a total of 1 unit of tetanus toxin in divided doses. In 3days the tone of the muscle improves and the tissue laxity around theeyes decreases.

This example illustrates that the administration of tetanus toxin tofacial muscle increases the tone and youthful appearance of the patient.A youthful appearance is due in part to good tone in facial muscles. Inthe aged this tone could be improved by administration (e.g., via directinjection) into the facial muscle. The muscles most likely to benefitfrom this treatment useful are the smile muscles rhizorius and quadratusand the periorbital muscles. Increased tone in these muscles flatten theredundant folds of skin seen in aging and are used largely as asubstitute for a blepharoplasty.

Example 11 Increased Muscle Mass as a Substitute for Exercise

A 25 year old weight lifter has 10 units of tetanus toxin injected intoboth biceps. In I-3 days the tone of the biceps muscles increases. In 1to 6 weeks the mass and strength of the muscle increases.

Increases in muscle tone and/or mass is generally desirable forcosmetic, competitive, preventative or rehabilitative reasons. Those whodesire the effects of exercise would undergo injection into the muscleof interest such as the biceps strictly for the purpose of increasingtone and causing hypertrophy. In competitive athletes the same methodcan be used for a functional effect. An example might be a weightlifterwith a relative weakness in certain arm muscles that cannot be correctedwith normal exercise and could be benited by undergoing a tetanus toxininjections. In certain conditions the effect of increased tone are bothcosmetic and medically desirable. One example is the muscle of theabdominal wall. Weak muscles of the abdominal wall allow sagging of theabdomen and also predispose the patient to back injury. Injection oftetanus toxin into the abdominal muscles increases the tone of theabdominal wall causing it to flatten and help with spinal alignment.

Example 12 Pharyngeal Paresis with Dysphagia

A 60 year old female suffering from dysphagia after a cerebrovascularaccident is injected with 1 unit of tetanus toxin into the inferior andmiddle constrictor muscles after 1-3 days the symptoms of dysphagiaimprove.

Example 13 Nasal Decongestion Nasal Mucosa

In this example, a 20 year old male with nasal congestion due toperenial allergic rhinitis is injected with 1 unit of tetanus toxin intothe mucosa covering each turbinate. After one week there is a noticeabledecrease in congestion.

Nasal congestion is the major symptom of allergic and infectiousrhinitis and is one of the most common complaints in all of medicine.The mucosa covering the intranasal turbinates is capable of changingthickness partly as a result of changes in blood flow. The mechanism ofdecongestion involves increased activity of the sympathetic nervoussystem. Specifically, increased tone in sympathetic nerves to the nasalmucosa contracts smooth muscle in arterioles and venules and shrinks themucosa.

This example illustrates that administration (e.g., injections) oftetanus toxin into the nasal mucosa causes nasal decongestion.

Example 14 Penile Erections and Ejaculation

A 40 year old male with impotence due to a diabetic neuropathy isinjected with 1 unit of tetanus toxin into the base of the penis causingincreased neural activity in the autonomic nerves as well as thepudendal motor nerves to the ischiocavernosis and bulbospongiosismuscle. In one week the patient can maintain an erection when aroused.

The control of the penile function is a complex mixture of bothparasympathetic and sympathetic innervation. Cholinergic sympatheticnerves from the sacral plexus cause the vasodilation enabling erections.Adrenergic sympathetic neurons activate the smooth muscle of the vasdeferens and seminal vesicles. Emission of ejaculate is a sympatheticresponse. Secretions of the bulbourethral and prostate are underparasympathetic control. Autonomic dysfunction from a variety of medicalreasons can cause impotence.

It is apparent from this example that the application of tetanus toxinto control the activity of the appropriate autonomic sympathetic nervesresults in the desired result.

Example 15 Increased Connective Tissue

A 70 year old female is injected with a total of 1 unit of tetanus toxindelivered in four separate injections to various quadrants of the skinof the face. Increased neural activity results in a thicker dermal layerto the skin and a more youthful appearance.

Connective tissue is formed by fibroblasts or myofibroblasts. It isknown that denervation of the motor and nerve supply to an area of skincauses a significant decrease in skin thickness. The activity of thenerve supply to skin apparently simulates the production of connectivetissue.

This example demonstrates that tetanus toxin can be applied (e.g., byinjection) to the dermal layer of the skin to increase connectivetissues, resulting in a more youthful appearance.

Example 16 Hair Loss

A fifty year old male with male pattern baldness is injected in the baldarea of the scalp with multiple injections of 0.25 unit of tetanustoxin. In one month the patient notes early hair regrowth in the area.

Hair loss appears to be in part due to decreased activity of theautonomic innervation to hair follicles. Experiments in rats show thatanagen (hair growth) is associated with increased sympathetic activitywithin the nerves surrounding the hair follicle. It is seen thatadministration (e.g., injections) of tetanus toxin into the skin inareas of hair loss increases autonomic activity and slows or reverseshair loss.

Example 17 Veterinary Uses of Tetanus Toxin

Cattle, goats, sheep, lamb, pigs, poultry, fish, invertebrates and otheranimals are all raised and harvested for their meat. In most cases theimportant meat harvested from these animals is muscle. Increased musclesize translates into increased meat production. In this embodimenttetanus toxin would be administered (e.g., injection) to animals tocause muscular hypertrophy.

In one example the pectoralis muscle of the turkey is increased byapplying an injection of 1 unit of tetanus toxoid into each pectoralismuscle of the turkey. After 2 days muscle tone would increase in themuscles and the mass of the muscle would increase.

A cow undergoes a lumbar puncture and 1 unit of tetanus toxin isinstilled into the cerebrospinal fluid. The next day the animal exhibitsmildly increased tone of all muscles. Over the next two months themuscle mass of the low increases by 10%.

Example 18 Increase in Milk Production

Milk is another product that is harvested from animals. Milk productionis largely hormonal but there is evidence that the nervous system playsa large role in secretion of milk and plays a role in production.

Direct administration (e.g., injection) into the mammary gland orretrograde administration (e.g., injection) through its duct can resultin increased neural activity and such increase translates into increasedmilk production.

A dairy cow has injected into each teat 1 unit of tetanus toxin. In 2days the tone of the smooth muscle within the teat increases and thequantity of milk produced increases.

Example 19 Sweating Skin

Sweating, also called hyperhydrosis, is under the control of thesympathetic nervous system but the neurotransmitter used by the postganglionic neuron is acetylcholine. The locations of clinical importantsweating include the armpit; the feet (the humidity causes the fungalinfection of athlete's foot); the genital area (crotch itch); the palmsand the brow.

In one example, a forty year male experiencing excessive sweating fromthe axillae is injected in this area with 1000 units of tetanus toxin.Decrease in sweating is noted within 3 days.

This example illustrates that an anticholinergic medication such astetanus toxin is capable of blocking the production of sweat.

Example 20 Rhinorrhea (Post Nasal Drip) Nasal Mucosa

Rhinorrhea is the production of excessive secretions from the nose andis a major symptom of allergic, infectious and vasomotor rhinitis.Anticholinergic medication is effective for blocking rhinorrhea forbrief periods or injected into the intranasal turbinates of humans. Theparasympathetic ganglia that contains the postganglionic cell bodiesthat supply the nasal secretory glands is in the pterygopalatineganglia.

In one example, a 70 year old female complains of profuse wateryrhinorrhea throughout the day. Injections of 500 units of tetanus toxinare made into both inferior turbinates with decreased rhinorrhea in 3days.

In another example, a 50 year old male complains of perennial allergiesand profuse nasal mucus rhinorrhea. A needle is passed from the oralside of the hard palate through the pterygopalatine canal and into thepterygopalatine space and 500 units of tetanus toxin are injected oneach side. The symptoms of rhinorrhea improve within 3 days fromtreatment with tetanus toxin.

Thus, blocking cholinergic neural transmission in the pterygopalatineganglia by using tetanus toxin is also effective at decreasingrhinorrhea.

Example 21 Prostatic Hypertrophy Prostate Gland

A 60 year old male with difficulty voiding due to prostatic hypertrophyis injected with 500 units of tetanus toxin into the prostate gland.Over the month following the injection with tetanus toxin the patientnotices gradual decrease in his symptoms.

Prostatic hypertrophy is common in males over 50 years of age and causesdifficulties in initiating urination. It has long been known thatdenervation of the autonomic innervation of the gland causes it todecrease in size.

This example demonstrates that injections of tetanus toxin into theprostate gland causes it to shrink.

Example 22 Asthma, COPD Pulmonary Mucus Secretion

A 60 year old male patient with bronchitis has the symptoms of excessivepulmonary mucus. 5000 units of tetanus toxin are mixed with 10 cc ofnormal saline and aerosolized and inhaled by the patient over a 30minute period. In 2 days the patient notes a decrease in mucusproduction.

A prominent symptom of many lung diseases is the production of excessiveamounts of mucus. These diseases include asthma, chronic obstructivepulmonary disease, bronchitis, bronchiectasis and cystic fibrosis.Pulmonary mucus is produced by small glands within the respiratorymucosa covering the bronchi.

This example illustrates that mucus production is controlled byapplication of tetanus toxin to inhibit parasympathetic neural activityof pulmonary mucosa.

Example 23 Asthma, COPD Bronchial Smooth Muscle

Many lung diseases have the symptom of chronic or acute airwayobstruction. The lumem of bronchioles is largely controlled bycontraction of smooth muscle that is under parasympathetic control.

A 13 year girl with asthma is placed under light anesthesia and abrochoscope is inserted trough the mouth and into the trachea. Using athin gauge transbronchial needle 20 injections of 100 units each oftetanus toxin are made through the mucosa. In the following weeks thesymptom of bronchospasm is improved. This example illustrates thattransmucosal absorption or injection of tetanus toxin through the mucosablocks parasympathetic activity and prevents bronchospasm.

Example 24 Salivation Parotid, Submaxillary and Sublingual Glands

Many neurologically impaired patients have difficulty preventing salivafrom entering their lungs. Contamination of the lungs with the bacterialaden saliva can lead to a lethal pneumonia.

A 60 year old female with amyotrophic lateral sclerosis has beenaspirating saliva. The patient has 100 units of tetanus toxin injectedinto each of the three major salivary glands bilaterally for a totaldose of 600 units. In two days salivation has decreased considerably andshe no longer aspirates saliva. Thus, salivation is underparasympathetic control and the amount of salivation has been shown todecrease when both animals and humans are injected with tetanus toxin.

Example 25 Sphincters Anal Fissures and Constipation

Increased tone in the anal sphincter causing constipation is calledoutlet obstruction and occurs in Parkinson disease as well as otherneurological conditions.

A 65 year old male patient with Parkinson's disease and associatedparadoxical activation of the puborectalis muscle during straining istreated with an injection of a total of 1000 units of tetanus toxin intotwo sites of the puborectalis muscle. Within 3 days the patientexperiences a decline in straining pressure during evacuation.

Chronic anal fissure is maintained by contraction of the internal analsphincter. Surgical sectioning of the sphincter is successful in 85% to95% of patients however permanently weakens the sphincter and may causeanal deformity and incontinence.

In another example, a 35 year old female with a chronic anal fissure isinjected with a total of 1000 units into two sites of the analsphincter. In 1-3 days tone of the anal sphincter decreases and thefissure heals over the following 2 months.

From these examples, it is apparent that injections of botulinumtoxincon successfully be used to relax the anal sphincter and allow itto heal.

Example 26 Achalasia Lower Esophageal Sphincter

Increased contraction tone of the cholinergic innervation of the loweresophageal sphincter can interfere with swallowing.

A 40 year old male has achalasia of the esophagus with severedifficulties in swallowing. A flexible endoscope is passed into theesophagus and 300 units of tetanus toxin are injected transmucossallyinto the lower esophageal sphincter. In 3 days the patient noticesimprovement in swallowing.

It is shown that this condition can be successfully treated withtransmucosal injections of tetanus toxin.

Example 27 Obesity Gastric Wall Muscle

A 30 year old female has a flexible endoscope passed through theesophagus and into the stomach. The antral wall muscle of the stomach isinjected with 1000 units of tetanus toxin. After 1 week the foodconsumption of the patient decreases and she begins to lose weight.

The feeling of hunger and satiation are partly related to the state ofcontraction of the stomach wall. The paralysis of the gastric antrumslows the emptying of the stomach and causes feeling of early satiety.It is therefore, shows that patients with morbid obesity can benefitfrom endoscopic injections of tetanus toxin into the stomach wall.

Example 28 Immune Tolerance

It has been shown that blocking the parasympathetic nerves to an area ofskin (or mucosa) followed by injection of antigen into the area invokesimmune tolerance to the antigen. Administration (e.g., injections) oftetanus toxin followed by the antigen can induce tolerance to theantigen. This effect can be used to treat or ameliorate autoimmunedisorders.

Autoimmune diseases with their putative antigenic proteins set forthparenthetically include without limitations: experimental autoimmuneencephelomyelitism (myelin basic protein); arthritis (type II collagen);uveitis (S-antigen, interphotoreceptor binding protein); diabetes(insulin, glutamine decarboxylase); myasthenia gravis (acetlylcholinereceptor); thyroiditis (thyroglobulin); and multiple sclerosis (myelin).

A forty year old female with multiple sclerosis is injected with 1000units of tetanus toxin into the skin of the left forearm. One week latermyelin is injected into the same site. Within a month the patientexhibits a decrease in symptoms.

Another area in which immune tolerance is beneficial is organtransplantation. Humans normally develop an immune reaction to theforeign proteins, especially major histocompatibility protein, that ispresent on the surface of the cells in the transplanted organ.

In another example pertaining to organ transplantation, a 40 year oldfemale requiring a kidney transplant is injected with 1000 units oftetanus toxin into the skin of the left arm. After one week cell urfaceantigens from a potential donor are injected into the same area. In onemonth the patient is tested for tolerance by injection of the same cellsurface antigens into the skin of the right forearm. No noticeablereaction indicates that tolerance has been achieved and the transplantcan be done.

Thus, administration of tetanus toxin into a region of (e.g.,injections) allows subsequent presentation of an antigen to invoketolerance to that antigen. This effect could be beneficial to patientswith autoimmune disease or potential recipients of allografts orxenograft organs.

Example 29 Gastric Acid

Gastric acid production is under the control of the parasympatheticnerves. However, an additional method of blocking the acid production isin the parietal cells that produce the acid. They secrete H₊ by vesiclerelease and can be blocked directly (Alexander et al., American Journalof Physiology, 273 (6 Pt. 2), F 1054-7, (1997)). A third method is toblock the hormones that increase acid production, secretin and gastrin.An additional beneficial effect would be to block the production of theenzyme trypsin.

Example 30 Holocrine Secretion

Holocrine glands are a class of skin secretory glands that produce alipid secretion and are partly under neural control. Holocrine glandsinclude sebaceous and follicular glands, cerumen glands and mammaryglands. Blocking the neural input to these glands by administration(e.g., injection) of tetanus toxin may be useful in a variety of medicalconditions, examples include acne where over production of secretion isthe basis for the inflammation and infection.

Also cerumen overproduction is one of the most common reasons for visitsto otolaryngologists. Administration (e.g., injection) to the skin ofthe ear canal would block cerumen production and prevent ear waxaccumulation in the ear canal.

In addition, the tetanus toxin may be used to block the activity of thesebaceous glands, thereby providing beneficial effects in the skincondition acne.

Example 31 Skin Disorders

Psoriasis, atopic dermatitis (hives), vitiligo are all related toparasympathetic activity. It has long been known that denervation of anarea of skin causes resolution of these skin disorders in the denervatedarea. Administration of the tetanus toxin (e.g., by injection) intoaffected areas can block the activity and cause improvement orresolution of the symptoms. Skin injections with tetanus toxin candecrease of the skin disorders listed above by decreasing theparasympathetic activity at selected sites.

Example 32 Migraine

Administration of the tetanus toxin (e.g., by injection) into thepterygopalatine ganglia would block postganglionic nerves to the carotidartery and thereby block the arterial spasms caused by these nerves thatis the basis for migraine headaches. Migraine like tension headachescould be treated by injection into the temporalis muscle.

Example 33 Adipose Tissue

Glucose uptake by adipose tissue is necessary for lipid production.Tetanus toxin delivered by liposomes blocks lipid glucose uptake by fatcells and can cause a decrease in their size. The cellular activity ofother endocrine cells can be inhibited by administration (via injection)of tetanus toxin. Endocrine cells affected by treatment with tetanustoxin include thyroid, pancreatic and tumor cells.

Example 34 Vomeronasal Organ

The vomeronasal organ senses pheromones and plays a role in reproductivebehavior and other autonomic drives. Administration (e.g., injection) oftetanus toxin into this organ can block, or increase these drives.

Example 35

Immune System T Cell Maturation and Release

Parasympathetic activity is related to maturation and release of T cellsfrom the thymus and spleen. T cells are the cellular mediators ofantigen recognition. Increased parasympathetic activity increases thematuration and release of T cells; the opposite occurs when these organsare acutely denervated. Administration (e.g., injection) of tetanustoxin into the thymus and/or spleen at various doses can either increaseor decrease the release of T cells.

A 25 year old patient with HIV and low T cell counts is injected with100-unit of tetanus into his thymus gland. After 1 week T cell levels inthe blood increase.

A 50 year old female with multiple sclerosis has an acute worsening ofher disease and is injected with 100 units into her thymus gland. Oneweek after the injection the symptoms are ameliorated.

Example 36 Sensory Uses of Tetanus Toxin

Unlike botulinum, tetanus toxin has been shown to bind and enter sensorynerves, undergo retrograde transport and cause anesthesia. Theseobservations have been made in experimental animals as well as inclinical tetanus. In addition to the decrease in afferent neuralactivity the tetanus toxin would block the release of inflammatorymediators from the sensory nerve (substance p, neuropeptide Y, CGRP) asthese neuropeptides are by SNARE mechanism using the protein VAMP thatis inactivated by tetanus toxin. The most important use for this effectcan be to block pain from any part of the body. For example, chronicjoint pain can be blocked by administration of the tetanus toxin (e.g.,by injection) into the joint bursa.

A 75 year old female with a degenerative right hip joint with chronicpain undergoes an injection of 1000 units of tetanus toxin into thejoint. Within one week the pain of the patient has decreased. The aboveexample illustrates that the specific target of administration can varywith a specific clinical condition but can include bone, cartilage,ligament, muscle, fascia, mucosa, skin, pleural membranes, epineurium,synovial membranes, neuromas, and smooth muscle.

Another use for sensory blockade is the axon-axonal reflexes underlyinginflammation. Sensory axons react to noxious stimuli by evoking a reflexvasodilation in the entire region innervated by the nerve (sometimesreferred to as the wheal and flair reaction). Tetanus toxin can blockthe release of the neuropeptides that evoke this reflex.

A 65 year old male with chronic bronchitis and the symptoms of excessmucus production and paraoxysmal coughing inhales an aerosolizedsolution of 1000 units of tetanus toxin for 30 minutes. Two days laterhis coughing decreases.

The above examples show that another use for sensory blockade can be inthe chronic cough, mucus production and bronchospasm initiated by intrapulmonary sensory receptors. In this embodiment the tetanus toxin isbest delivered by inhaled aerosol.

Example 37 Cardiovascular System

The cardiovascular system, the heart, arteries, and veins have extensiveautonomic innervation. In the heart sympathetic activity causesincreased heart rate and contractile force. Parasympathetic stimulationslows the heart and decreases contractile force. The rate of cardiaccontraction is controlled by the sinoatrial node, a small ganglia in theright atrium of the heart, while the propagation of the contraction fromatria to ventricles is controlled by the atrioventricular node (AV)node.

Cardiac disorders which can be treated with tetanus toxin pharmaceuticalformulations include the cardiac arrythmias: tachycardia, bradycardia,and ventricular fibrillation. Additional disorders include coronaryspasm resulting in angina and/or myocardial infarction. Catherization ofthe coronary arteries allows release of tetanus toxin into the bloodperfusing the ventricles and the particular coronary artery used allowssome localized distribution of the toxin to areas of the heart mostaffected. In these cases inhibitory doses of tetanus toxin can decreasecardiac excitability by inhibiting sympathetic stimulation and by adirect effect on cardiac myocytes. Injection of tetanus toxin intocoronary arteries at the higher inhibitory doses results in decreasedsympathetic activation of the smooth muscle and decreases the intensityof coronary artery spasm ameliorating angina. Injection of tetanus toxininto the sinoatrial (SA) node at excitatory levels can increaseparasympathetic activity and slow the heart rate decreasing thepossibility of arrythmia and/or angina. The use of catherizationtechniques that reach the above mentioned areas of the heart are wellknown to those skilled in the art and do not require undueexperimentation.

Example 38 White Blood Cells

Monocytes and macrophages have receptors that allow uptake andinternalization of tetanus toxin. Once internalized the toxin disruptsthe molecular mechanism underlying cellular mobility as well assecretion of vesicles. Many inflammatory processes are associated withthe migration of macrophages or to the area. Once in the region ofinflammation these cells release other cytokines that increaseinflammation or cause tissue breakdown. Administration of tetanus toxincan slow or stop the migration of macrophages as well as prevent therelease of inflammatory mediators from cells in the area. Tetanus toxinis also capable of blocking the aggregation and secretion of neutrophilsbut requires of vector like liposomes for cell internalization.Rheumatoid arthritis is an example of a disorder that can benefit fromthis therapy, with administration directly into the synovial bursa. Thisis an example of using tetanus toxin to control cellular activity ofspecific target cells such as macrophages.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will appreciate that other and further modifications and changes canbe made without departing from the true spirit of the invention, and itis intended to include all further and other such modifications andchanges which come within the scope of the invention as set forth in theappended claims.

1. A method of administering a therapeutically effective amount of tetanus toxin to a selected site, having target neurons whose activity is increased thereby, of an animal in need of increased activity of said neurons, wherein the said therapeutically effective amount of tetanus toxin causes an increase in neural activity or an excitatory response of the neural activity at the selected site.
 2. The method of claim 1, comprising administering to a selected site affecting target neurons of an animal an amount of tetanus toxin sufficient to cause an excitatory response of the nerve function at the selected site affected by the target neurons.
 3. The method of claim 2, wherein the therapeutically effective amount is from about 0.001 units to about 2000 units for the selected site.
 4. The method of claim 3, wherein the therapeutically effective amount is from about 2 units to about 4 units for the selected site.
 5. The method of claim 2, wherein the selected site comprises tissues or organs affected by sleep apnea and snoring, dysphagia, scoliosis, strabismus, muscle atrophy, tissues or organs affecting salivary gland secretions, vaginal secretions, lacrimal secretions, mammary secretions tissues or organs affected by nasal congestion, impotence, hair loss, connective tissue of lax, aged skin or hypotension and neurologically impaired muscles affected by muscular dystrophy, amyotrophic lateral sclerosis or myasthenia gravis, decrease in muscle mass, or decrease in facial muscle tone.
 6. The method of claim 2, wherein the selected site comprises vascular, pulmonary, genitourinary or gastrointestinal smooth muscle, the lower esophageal sphincter, anal sphincter, bladder wall, bladder and urethral sphincter, vaginal sphincter, stomach wall, pyloric sphincter, upper esophageal sphincter, and colon wall muscles.
 7. The method of claim 5, wherein the organs affecting saliva production include submandibular gland, parotid gland, sublingual gland, or minor salivary glands of the oral mucosa.
 8. The method of claim 1 wherein a fragment of tetanus toxin is used alone, where that fragment is the light chain of tetanus toxin or the fragment is combined with a second protein or other molecule or delivered via liposomes.
 9. The method of claim 1 wherein the therapeutically effective amount of tetanus toxin causes a modulation of neural activity at a site other than the selected site.
 10. The method of claim 9 where the selected site is the vomeronasal organ.
 11. The method of claim 1 wherein the therapeutically effective amount of tetanus toxin invokes immune tolerance to autoimmune antigens or antigens of a transplanted organ. 